The CA protein is the major structural protein in the retrovirus core and a contributor to several stages of the virus life-cycle. In the mature virion core, CA forms a protein shell that surrounds the ribonucleoprotein complex of viral RNA and replication enzymes. The integrity of the CA protein and the shell that it forms are critical for the ability of the virus to replicate its genome and to establish infection in the target cell. As such, the CA protein and the maturation process that creates the functional capsid are attractive targets for antiviral intervention. However, the limited knowledge about the precise molecular details of these events seriously limits the ability to exploit these targets. Once CA is cleaved from its polyprotein precursor, CA undergoes a complicated choreography of conformational changes and intermolecular interactions to create the functional capsid. We have developed an in vitro assay for CA assembly that faithfully recapitulates many features of capsid assembly in situ, including the formation of capsid-like shells that contain CA in both hexameric and pentameric arrangements. We have demonstrated that CA assembly in vitro is a well-behaved nucleation-driven process that is highly amenable to the study of the genetic and biochemical factors that control the nucleation of capsid assembly. We have also produced a high resolution structural model for the CA protein in a pentameric arrangement that both identifies novel intermolecular interactions between the CA subunits and provides a testable model for the control of capsid nucleation. In the proposed studies we will test key features of this model, including the action of particular interfacial residues, of the ?8 helix and the ?8-??9 loop, and of the ?-hairpin and flexible outer loop regions of the N-terminal domain in controlling the nucleation of capsid assembly. We will seek to define CA intersubunit interactions in capsid assembly intermediates and the role of other virion constituents in the process. Achieving these goals will be a major step toward understanding one of the most mysterious aspects of the retroviral life cycle and the ultimate identification of new strategies for blocking the maturation events and thereby the replication of the virus. PUBLIC HEALTH RELEVANCE: In all retroviruses, including the Rous sarcoma virus and the human pathogens HIV and the human T-cell lymphotropic virus, a stage of the virus life cycle known as maturation involves very dramatic structural changes in the interior of the virus particle, leading to the activation of its infectious potential. The studies described in this proposal will use a combination of genetic and protein structural approaches to examine the molecular mechanisms that control this process. A detailed understanding of this essential step of virus infection will allow development of better maturation inhibitors for use as antiretroviral drugs.